Bioscience Reports 2, 675-685 (1982)
Printed in Great Britain
675
3 ' , 5 ' - C y c l i c - n u c l e o t i d e - d e p e n d e n t p r o t e i n k i n a s e of
s q u a m o u s cell c a r c i n o m a of t h e p r o s t a t e
R. VI3AYVARGIYA and R. L. SINGHAL
Department of Pharmacology, Faculty of Health Sciences,
University of Ottawa, Ottawa, Canada
(Received 1 August 1982)
An adenosine 3',5'-cyclic-monophosphate(cyclic AMP)d e p e n d e n t p r o t e i n kinase has been i d e n t i f i e d and
partially purified from the rat prostate tumor induced
by 20-methylcholanthrene.
This enzyme is stimulated
2- tO 3-fold by the nucleotide. Equilibrium studies at
pH 5.0 suggest the presence of a major class of binding
site for cyclic AMP with an association constant of
approximately 108 M-l.
The concentration of binding
s i t e is about I pmol/mg of protein of the enzyme
preparation.
The enzyme is stimulated by other cyclic
nucleotides as well9 but only by higher concentrations.
In c o m p a r i n g the a b i l i t y of d i f f e r e n t histone subfractions9 casein and protamin% to serve as substrate
for this particular protein kinase, maximal cyclic-AMPdependent enzyme activity was observed with histones.
The r e s u l t s suggest that factors contributing to the
malignant growth of the prostatic tissue do not directly
i n v o l v e c h a n g e s in the characteristics of a cyclicAMP-dependent protein kinase.
It is known that steroid hormones exert at least some of their
diverse metabolic effects on target organs through the stimulation of
the adenylate cyclase-cyclic AMP system.
5inghal et al. (1) and
Sutherland and Singhal (2) demonstrated that administration of male
sex h o r m o n e s to a n d r o g e n - d e p r i v e d rats p r o d u c e s a significant
stimulation of prostatic adenylate cyclase.
Further studies revealed
that t r e a t m e n t with exogenous cyclic AMP exhibits an 'andromimetic'
action on male sex accessory organs (314).
Investigations on the
mechanisms by which cyclic AMP exerts its various metabolic effects
have brought to light the importance of protein kinase in its action
(5~6).
In order to understand the role of protein kinase in the
mechanism of action of the cyclic nucleotid% protein kinase from
s e v e r a l t i s s u e s has been purified and characterized (6-9).
Such
studies have demonstrated the existence of cyclic-AMP-dependent and
-independent forms of protein kinases in a variety of tissues.
The
p r e p a r a t i o n and characterization of a cyclic-AMP-dependent protein
kinase isolated from canine prostate was earlier reported by Tsang and
Singhal (10).
Since cyclic AMP is reported to be a modulator of
growth during malignant tumor formation (11), it became of interest
to isolate and characterize the nature of protein kinase from rat
prostatic tumor.
The present paper incorporates our observations of
9
The Biochemical Society
676
VI3AYVARGIYA & SINGHAL
t h e i s o l a t i o n , p u r i f i c a t i o n , and c h a r a c t e r i s t i c s of a cyclic-AMPd e p e n d e n t p r o t e i n kinase from the p r o s t a t i c t u m o r induced by
20-methylcholanthrene.
M a t e r i a l s and Methods
[32p]ATP (13.1 Ci/mmol) and OmnKluor were obtained from New
England Nuclear Corporation, Boston, Mass., and cyclic [3H]AMP (27
Ci/mmol) was purchased from Amersham Searle, Arlington Heights,
III.
c~-Casein, protamine, histone mixture (Type I I A ) , DEAE-cellulose,
and all unlabelled nucleotides were obtained from Sigma Chemical Co.,
St. Louis, Mo.~ and the histone subfractions were purchased from
Worthington Biochemical Corp., Freehold, N.3.
The prostatic tumor
was originally induced with 20-methylcholanthrene.
Tumors were implanted in male host rats using a 14-gauge trochar.
Tissue which had previously been frozen and stored at -70~
was
thawed quickly and suspended in Hank's medium 199 ( i g of tissue/3
rnl medium 199).
0.I ml of tissue suspension was implanted subcutaneously in male F3t~4 rats that were 40-50 days old.
Control
animals received an injection of Hank's medium 199 alone (0.3 ml)
and were sacrificed at 60 days of age. Animals were maintained on
Purina Rat Chow and water ad libitum, and were housed in a room
under controlled lighting conditions.
Preparation of protein kinase
C y c l i c - A M P - d e p e n d e n t p r o t e i n kinase was partially purified by
m o d i f i c a t i o n of r e p o r t e d procedures (5~12,13).
The tumor tissue
excised from host rats (totalling about 70 g) was homogenized in 3
vol. of buffer (0.05 M Tris/HCl, pH 7.4, 0.25 M sucrose, 0.05 M KC1,
5 mM MgCI2) and the homogenate centrifuged at 650 9 for 30 min.
The resultant supernatant containing the soluble cyclic-AMP-dependent
protein kinase was then subjected to precipitation at pH 5.5 and then
centrifuged at 4000 9 for 15 min. The supernatant was again adjusted
to pH 7.4 and subjected to (NH~)2SO ~ (0.32 g/ml) precipitation. The
pellet formed from centrKugation at 4000 g for 15 min was dissolved
in a volume of buffer (0.01 M T r i s / H C l - 1 mM 2-mercaptoethanol~
pH 7.4) e q u i v a l e n t to a p p r o x i m a t e l y one-fifth of that used for
homogenization of the tissue and dialyzed overnight against 3 vol.
(7 litres each) of the same buffer. The solution was then centrifuged
at 10 000 g for l0 min to remove any precipitate, and was applied to
a DEAE-cellulose column previously equilibrated with the buffer just
before loading. The column, after washing with the buffer again (2 x
the void volume of the column) was subsequently eluted with a linear
NaC1 gradient (0.05 M - 0.35 M) containing 0.01 M Tris/HCt - 1 mM
2-mercaptoethanol, pH 7.4. Fractions comprising both peaks exhibiting
cyclic AMP binding and cyclic-AMP-dependent kinase (peak I, fractions
5 and 6, and peak II, fractions 16-23) were pooled separately, dialyzed
against the column buffer without NaC1, and stored in small portions
at -20~
Data presented in this paper describe the characteristics of
t h e t u m o r protein kinase Muted at 0.19 M NaCI (peak II) during
purification on DEAE-cellulose.
PROTEIN KINASE OF SQUAMOUS CELL CARCINOMA
Protein
kinase
677
assay
Protein kinase was assayed by measuring the incorporation of 32p
into histone mixture (Type IIA) by modification of the method of
Sanborn et al. (12).
The assay mixture (final vol. 0.13 ml, pH 6.5)
contained 46 mM sodium acetat% 1.5 mM sodium fluorid% 3.1 mM
l:heophylline, 0.24 mg of histone mixture~ 17 mM MgCl2~ 85 pM ATP~
67 pg of enzyme preparation (Peak II) after DEAE-cellulose step, and
L~.6 pM cyclic AMP (for measuring the activity of the cyclic-AMPdependent enzyme). Immediately after the addition of the enzyme to
the incubation mixtur% the reaction was initiated by the addition of
[y-32p]ATP-MgC12 (200 000 c.p.m.) and incubated at 30~ for 7 min.
The reaction was terminated by the addition of 5 ml of 20% tric h l o r o a c e t i c acid.
The protein precipitate was then separated by
filtering on a Millipore filter (HAWP 0.45 pM) and the radioactivivty
c o u n t e d in i0 ml of scintillation 'cocktail' containing 7 parts of
Omnifluor solution (4 g of Omnifluor per litre of scintillation-grade
toluene) to 3 parts of ethylene glycol monoethyl ether.
Appropriate
blanks without histone and enzyme preparation were run simultaneously
with the experimental samples.
Cyclic AMP binding assay
This was c a r r i e d out in a reaction mixture containing 0.05 M
sodium acetate) 1 mM EDTA, pH 5.07 in a final vol. of 250 pl as per
the modified method of Sanborn et al. (14).
50 pl of the enzyme
preparation (Peak II) after DEAE-cellulose purification was used with
2.4 nM cyclic [3H]AMP. After incubation at 0~ for 3 h) the assay
mixture was passed through a Millipore filter (HAWP 0.45 pM) which
was then counted for radioactivity in 10 ml of scintillation cocktail as
described for the protein kinase assay. Protein was determined by the
m e t h o d of L o w r y et al. (15) with bovine serum albumin as the
standard.
Results
Preparation of cyclic-AMP-dependent protein kinase
I s o l a t i o n and p a r t i a l p u r i f i c a t i o n of diethylamino cellulose of
cyclic-AMP-dependent protein kinase from tumor tissue yielded two
peaks ( f r a c t i o n s I and II~ Fig. i)~ both of which demonstrated
c a t a l y t i c and cyclic-AMP-binding activities (Table 1). The activity of
t h e major peak ( f r a c t i o n II) which eluted at 0.19 M NaCl was
stimulated 3-fold by 5 pM cyclic AMP. The minor peak (fraction I)
eluted at 0.09 M NaCI and the activity of this fraction was doubled
by the addition of the cyclic nucleotide.
Effect of various concentrations of cyclic AMP
The relationship between protein kinase activity and cyclic AMP
concentration was also studied (Fig. 2).
Whereas a concentration of
10 mM cyclic AMP was necessary for half maximal velocity~ maximal
678
VI3AYVARGIYA & SINGHAL
400 ~ ~cO
tl.
3oo O ~8
9
200 ~ ~
-o
100 o')
--<
::1.0
o
k
z4f-
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15 20 25 30 35
FRACTION NUMBER
i
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O
Fig. 1.
Chromatographic pattern of rat prostatic
squamous-tumor protein kinase and cyclic-AMP-binding
activities.
The enzyme (0.3 g of protein) was
applied to a DEAE-cellulose column (16 cm x 30 cm)
and eluted with a linear gradient of 0.05-0.35 M
NaCI solution (200 ml of each) after extensive
washing with 0 , 0 5 M NaCI. The buffer contained 0.01
M Tris/HCl and 1 mM 2-mercaptoethanol, pH 7.4.
Units
are expressed as c.p.m./sample. Q, 32p
incorporated into histone mixture in the presence of
4.6 ~M cyclic AMP without correction for sample
blank.
O, Cyclic [3H]AMP bound - concentration of
NaCI in elution buffer. Proteins wre estimated in a
50-~g aliquot of fractions collected.
k i n a s e a c t i v i t y o c c u r r e d w h e n t h e c y c l i c AMP c o n c e n t r a t i o n in t h e
incubation
medium
w a s 7.5 gM,
F u r t h e r i n c r e a s e s in t h e c y c l i c
n u c l e o t i d e c o n c e n t r a t i o n r e s u l t e d in a p r o g r e s s i v e l y d e c r e a s e d s t i m u l a t i o n of t h e t u m o r t i s s u e e n z y m e a c t i v i t y ,
PROTEIN KINASE OF SQUAMOUS CELL CARCINOMA
Table io
679
Purification of cyclic-AMP-binding and cyclic-AMP-dependent
protein kinase activities from rat prostate tumor
Standard conditions~ as described in the text~ were
activities of cyclic-AMP-binding and cyclic-AMP-dependent
Protein kinase activity
(pmol 32p incorp./7 min)
Fraction
Total
Per mg protein
used for assaying
protein kinase.
cAMP-binding capacity
(pmol [3H]cAMP bound/3 h)
Total
Per mg protein
I High-speed-centrifugation
supernatant
39
4.1
x 102
0.26
2.74
II pH precipitation
65
7.64 x 102
0.20
2.35
Ill (NH4)2SO 4
precipitation
103
1.37 x 103
0.54
7.2
IV Column eluate
135
2.01 x 103
0.52
7.76
200
~
160
~
140
~
~O
120
100
Z
8O
O~
60
Q 40
O
E
the
20
._J_~\ I
I
I
l
I
I
I
I
0 11 10 9
8 7 6
5 4
CYCLIC AMP C O N C E N T R A T I O N (-LOG M)
Fig.
2.
Effect
of 3'95'-cyclic AMP on protein
kinase
activity
in rat prostate tumor.
Protein
kinase
activity
is e x p r e s s e d
as pmol
of 32p
incorporated/mg
protein in the presence of 1 ~M
cyclic AMP.
Assays were carried out under standard
conditions
as described
in the text~ except for
variations
in the concentration
of the indicated
cyclic nucleotide.
6g0
VI3AYVARGIYA & SINGHAL
Equilibrium studies on cyclic AMP binding
The association of cyclic A M P with the protein kinase was studied
to further characterize the molecuJar interaction of this cyclic
nucleotide with its dependent protein kinase.
A Scatchard plot
analysis (16) of the binding data (Fig. 3) suggested noncooperativity
and one order of cyclic-AMP-binding sites.
The association constant
(/<assoc.) at 0~ and pH 5.0 was approximately 1.37 x 109 M-I and
the concentration of binding sites was l.l pmol/mg enzyme protein.
S-
:E
<
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._,,.I
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I.,L_ O,.
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7_
12)
.6
,5
'........
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_
0
0
"",,.,Q.,
"-, ,,,
.Z
I---
<
.1
I
I
I
;
I
i
I
t
i
i
.!
.Z
,3
.4
.S
.6
.7
.8
.9
1,0
10-9x BOUND CYCLIC AMP (mmol/mg
"'":~..
1.1
o f PROTEIN)
Fig. 3.
Scatchard plot describing the interaction
at e q u i l i b r i u m
between prostatic cyclic-AMPdependent
protein
kinase
and c y c l i c AMP.
Cyclic-AMP-binding
activity was assayed in an
incubation medium (final vol. 0.25 ml~ pH 5.0)
containing 2.4 nM cyclic [3H]AMP, 0-72 nM unlabelled
cyclic AMP, 0.25 M sodium acetate, 1 mM EDTA, and
I00 Ng of protein of Fraction II, enzyme preparation
after DEAE-cellulose chromatography.
Incubation was
carried out at 0~ for 3 h and protein-bound cyclic
AMP was separated by Millipore filtration as
described in the text. Zassoc. is 1.37 x 109 M -I
and the concentration of the binding site is 18
pmol/mg of protein.
PROTEIN KINASE OF SQUAMOUS CELL CARCINOMA
681
Nucieotide stimulation of protein kinase
The
ability
of
various
3',5'-cyclic mononucJeotides
to
stimulate
tumor tissue protein kinase is illustrated in Fig. #.
Cyclic AMP was
markedly more e f f e c t i v e at lower concentrations than any of the other
nucleotides tested.
Whereas cyclic AMP produced maximal stimulation
of the enzyme at 7.5 pM concentration, cyclic UMP, the post potent
analogue tested, only elicited 55% of maximal activity at an equimolar
concentration.
Cyclic UMP and cyclic GMP were able to stimulate
protein kinase activity to levels similar to those obtained with 7.5 pM
100 -
v
80
[ 4t~---t
= =
: -"
C o
cAMP
cUMP
cCMP
cTMP
db cAMP
e/
/
/
~'~
~'~
/
/
I.->
I--
u
<
60-
D []
lit
z
G
40-
O
v
2O
I
11
-log[CYCLIC
i
I
I
i
i
I
9
10
9
8
7
6
5
4
NUCLEOTIDE CONCENTRATION
(M)]
Fig. 4.
Effect of various 3',5'-cyclic mononucleotides on rat prostatic squamous-cell-carcinoma
protein kinase activities.
Kinase activity is
expressed as a percentage of that observed in the
presence of i pM cyclic AMP.
Assays were carried
out under standard conditions as described in the
text, except for variations in the concentration of
the indicated cyclic nucleotide.
The various
3',5'-cyclic mononucleotides tested are shown on the
figure.
682
VI3AYVARGIYA & SINGHAL
cyclic AMP; however, the other cyclic nucleotides (dibutyryl cyclic
AMP, cyclic TMP, and cyclic CMP) appeared to have less stimulatory
potential on kinase activity.
Ability of various proteins
to serve as substrate
Cyclic AMP (it.6 pM) markedly enhanced 32p phosphorylation of
calf thymus histones, particularly types IIA, F2b, and F3. Cyclic AMP
s t i m u l a t i o n of the e n z y m e a c t i v i t y on the F2b histones at any
concentration tested was more marked than any of the other substrates tested.
The optimal concentration of each histone protein
which resulted in maximal 32p transfer to the substrate did not appear
to coincide with maximal stimulation of the enzyme by cyclic AMP, as
measured by the percent increase from respective controls.
Discussion
P r o t e i n kinases from s e v e r a l t i s s u e s have been isolated and
characterized (6-8,17). Cyclic-AMP-dependent protein kinase has been
d e t e c t e d in a v a r i e t y of tissues including rat prostate ( t 8 - 2 1 ) .
D e s p i t e v a r i a t i o n s in the assay conditions employed by different
workers, some similarity seems to exist in protein kinases obtained
from various sources.
It has been possible to identify two cyclicAMP-dependent protein kinases from squamous prostatic carcinoma of
rats.
Other investigators have shown the presence of multi-forms of
c y c l i c - A M P - d e p e n d e n t p r o t e i n kinases in a v a r i e t y of t i s s u e s
(10,21-25).
Although a cyclic-AMP-binding protein void of kinase
a c t i v i t y is not thus far r e p o r t e d , cyclic-AMP-independent kinase
activity has occasionally been found. The properties of protein kinase
of the tumor isolated from the major peak eluted from the chromatographic column with 0.19 M NaCI compares favorably with the enzyme
isolated from rat testicular interstitial cells (26) and the dog prostate
(10). Studies on the ability of various cyclic nucleotides to stimulate
protein kinase isolated from rat tumor showed that cyclic AMP is
capable of stimulating the enzyme several-fold as compared to the
e f f e c t produced by other nucleotides. Maximal activity of the enzyme
was observed with a cyclic AMP concentration of about 2.5 x 10-6 M,
half maximal velocity occured at about 10-s M concentration. Of all
the cyclic nucleotides tested, cyclic CMP was found to be the least
effective in this respect.
Cyclic UMP and cyclic GMP could produce
c o m p a r a b l e a c t i v a t i o n only in much higher concentrations.
Even
dibutyryl cyclic AMP, the lipid-soluble analogue of cyclic AMP, was
found to be less active than the parent nucleotide. It was found to
stimulate kinase activity only to the extent of 65% of that obtained
with optimal cyclic AMP concentration.
The cyclic AMP dependence of the enzymes suggests the presence
of high affinity for the nucleotide in squamous prostatic carcinoma of
rats. Our data indicate a Z<assoc. = 1.4 x 109 M-1 (pH 5.0, 0~
and
a concentration of binding site of 1.1 pmol/mg of enzyme protein.
Using i d e n t i c a l e x p e r i m e n t a l c o n d i t i o n s , Tsang and Singhal (10)
reported a Kassoe. = 0.78 x 108 M-1 for protein kinase of the normal
canine prostate, and Sanborn et al. (12) found a Kassoc. of 0.6 x 108
M-1 - 1.01 x 108 M-I for the same enzyme in bovine endometrium.
]PROTEIN KINASE OF $QUAMOUS CELL CARCINOMA
683
"These values differ to some extent from those of Shima et al. (20),
who reported a Kassoc. of 0.2 x 108 M-I for cyclic A M P with respect
to a protein kinase from rat prostatic sarcoma tissue. The variations
r ep o r ted in the values of Kassoc. may be due to the differences in
assay conditions and/or the duration of the growth period of the tumor
(when excised) and, thus, the degree of necrosis.
The substrate affinity of the isolated protein kinase was studied
using a variety of substrates.
The enzyme was found to have a
g r e a t e r affinity for histones as compared to a casein and protamine.
This property of the enzyme is similar to the protein kinase obtained
from adipose tissue (27), brain (28), bovine thyroid (29), and heart
(18).
In contrast, protein kinase isolated from lobsters and fish (30)
has been reported to have greater substrate activity for casein.
It is reported that prostatic tumor tissue (20) and transformed cell
('.31) contain lower amounts of cyclic AMP in comparison to those
present in normal cells.
It would t h e r e f o r e appear that the prostatic
protein kinase should have increased affinity for this cyclic nucleotide
or a g r ea t e r number of binding sites if the enzyme has a role in
i n c r e a s e d cell proliferation.
Our data indicate that the Km with
r es p ect to cyclic AMP and the affinity of this cyclic nucleotide to the
p r o t e i n kinase enzyme was not altered in squamous prostatic carcinoma.
Even the concentration of the binding site was found to be
lower than that present in other tissues.
The c h a r a c t e r i s t i c s of the
cyclic-AMP-dependent protein kinase isolated from the prostatic tumor
thus suggest that changes in the mitotic activity of the prostatic
tissue do not directly involve the cyclic-AMP-dependent protein kinase
en zy me to phosphorylate proteins.
Acknowledgements
This i n v e s t i g a t i o n was supported by a grant from the Ontario
Cancer Treatment and Research Foundation. Dr. R. Vijayvargiya is a
Visiting Scientist from the Medical College, Indore (M.D.), India.
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VI3AYVARGIYA & SINGHAL
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PROTEIN KINASE OF SQUAMOUS CELL CARCINOMA
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